Molecular Mechanism Of Moxidectin Inducing Autophagy Arrest In Colorectal Cancer Cells

Research background and purpose:Colorectal cancer is a common malignant tumor of digestive tract,with high morbidity and mortality.Traditional treatment is not effective,so it is urgent to find new therapeutic drugs for colorectal cancer.Recent studies have shown that Macrolides have anti-tumor activity,and Moxidectin can inhibit glioblastoma through autophagy and apoptosis pathways,suggesting that Moxidectin may also have a good anti-tumor effect on colorectal cancer.Therefore,to investigate the effect and specific molecular mechanism of moxidectin on colorectal cancer is helpful to optimize its clinical application in the treatment of colorectal cancer.Materials and Methods:1.Effect of Moxidectin on the proliferation of CRC cells(1)MTT and cell clonal formation experiments to determine the effect of Moxidectin on the proliferation of CRC cells.(2)BALB/c Nude mouse CRC HCT15 cell subcutaneous tumor model was established to evaluate the inhibitory effect of Moxidectin on the proliferation of CRC cells at the animal level.2.Study on the mechanism of Moxidectin inhibiting CRC proliferation(1)Lactate dehydrogenase(LDH)release experiment to study the necrotic effect of moxidectin on CRC cells.(2)Western blotting and Annexin V-FITC/PI double staining were used to study the apoptosis of CRC cells by Moxidectin.(3)Western blotting was used to study the effect of Moxidectin on autophagy related proteins in CRC cells.(4)Transmission electron microscopy,immunofluorescence and m RFP-GFP-LC3tandem fluorescence were used to determine the effect of Moxidectin on the autophagy level of CRC cells.3.Moxidectin inhibits CRC cell proliferation through autophagy(1)Western blotting was used to study the effects of autophagy intervention drugs and moxidectin on autophagy related proteins in CRC cells.(2)MTT and cell cloning experiments were conducted to clarify the effect of autophagy intervention on Moxidectin s inhibition of CRC cell proliferation.4.Moxidectin inhibits AKT/m TOR signaling(1)High-throughput sequencing and Real-time Quantitative polymerase chain reaction(RT-PCR)were used to investigate whether Moxidectin mediated endoplasmic reticulum stress in CRC cells.(2)Western blotting,the effect of moxidectin on AKT/m TOR signaling pathway related proteins in CRC cells.(3)MTT and cell cloning experiments were conducted to clarify the effect of Moxidectin on the inhibition of CRC cell proliferation by interfering with AKT/m TOR signaling pathway.5.Study on the intervention of Moxidectin in the expression of Cystic Fibrosis Transmembrane Conductance Regulator(CFTR)(1)RT-PCR to explore the effect of moxidectin on CFTR expression in CRC cells.(2)The Cancer Genome Atlas(TCGA)database analyzed the relationship between CFTR and CRC.(3)Western blotting to clarify the effect of CFTR intervention on Moxidectin promoting CRC autophagy related proteins.(4)MTT and cell cloning experiments were conducted to clarify the effect of CFTR intervention on Moxidectin inhibition of CRC cell proliferation.Result:1.Moxidectin inhibits the proliferation of colorectal cancer cells.1.1 Moxidectin inhibits the proliferation of colorectal cancer cells in vitroAfter treatment with Moxidectin for 24 h and 36 h,the survival of colorectal cancer cells in HCT15,HCT116,DLD-1 and SW620 cells was not significantly affected at low concentration.With the increase of drug concentration and administration time,the survival rate of tumor cells decreased significantly.Normal colorectal epithelial cells NCM460 treated with Moxidectin below 16μmol/L has no significant proliferation inhibition.Finally HCT15 and SW620 cells were finally selected.The administration time was 36 h,and the administration concentrations were 4μmol/L,8μmol/L and 12μmol/L of moxidectin to carry out follow-up experiments.The clonal formation experiment showed that the number of cell clones of colorectal cancer HCT15 and SW620 cells treated with 4μmol/L,8μmol/L and 12μmol/L moxidectin decreased significantly compared with the control group in a concentration-dependent manner.1.2 Moxidectin inhibited the development of subcutaneous colorectal cancer in nude miceHuman colorectal cancer HCT15 cells were injected subcutaneously into nude mice to construct a subcutaneous tumor model.The tumor volume was 75mm~3and was treated with Moxidectin.The results showed that compared with the control group,Moxidectin treatment group significantly reduced tumor volume and weight,and had no obvious toxicity to mice.2.Study on the mechanism of Moxidectin inhibiting the proliferation of colorectal cancer2.1 Moxidectin have no significant effect on apoptosis and necrosis of colorectal cancer cellsAfter Moxidectin treatment of colorectal cancer HCT15 and SW620 cells,the results of lactate dehydrogenase release experiment showed that Moxidectin had no significant effect on the induction of colorectal cancer cell necrosis,which was not statistically significant.Western blot detection of apoptosis marker protein Cleved-Caspase3 showed that Moxidectin had no significant activation effect on apoptosis-related proteins,with no statistical significance.Flow cytometry with Annexin V/PI double staining showed that Moxidectin did not induce apoptosis in colorectal cancer cells,with no statistical significance.2.2 Effects of moxidectin on autophagy of colorectal cancer cellsMoxidectin was used to treat colorectal cancer HCT15 and SW620 cells,and western blotting results showed that the proportion of autophagy marker protein LC3II/I was increased after treatment for 24 h and 36 h.Transmission electron microscopy(TEM)showed that Moxidectin promoted the accumulation of autophagic vesicles in colorectal cancer cells compared with untreated control group.Autophagy associated protein LC3 was detected by immunofluorescence assay,and the results showed that moxidectin could significantly increase the expression of LC3II in colorectal cancer cells.m RFP-GFP-LC3 tandem fluorescence assay showed that the expression of LC3was increased and the degradation was decreased by Moxidectin compared with the control group.Western blotting indicated that Beclin1,p62 and LC3II/LC3I ratio were increased.3.Moxidectin inhibits colorectal cancer cell proliferation through autophagyColorectal cancer HCT15 and SW620 cells were treated with 3-methyladenine(3-MA)and Moxidectin alone or in combination.Western blots showed that the combination of 3-MA and moxidectin reduced the ratio of LC3II/LC3I and showed no significant difference in p62 expression compared with moxidectin group.MTT and clonal formation experiments showed that 3-MA combined with Moxidectin significantly increased cell proliferation compared with Moxidectin experimental group.Hydroxychloroquine(HCQ)and moxidectin alone or in combination were used to treat colorectal cancer HCT15 and SW620 cells.Western blot results showed that the combination of HCQ and Moxidectin reduced the LC3II/LC3I ratio,and MTT and clonal formation assay showed that the combination of HCQ and Moxidectin significantly inhibited cell proliferation compared with Moxidectin treatment group.4.Moxidectin inhibits AKT/m TOR signaling pathway in colorectal cancer cellsMoxidectin was used to treat colorectal cancer HCT15 and SW620 cells,and RT-PCR showed that Moxidectin increased the expression of GPR78 at the m RNA level.Western blotting showed no significant differences in the protein expressions of AKT,P70S6K and m TOR,but the phosphorylation levels decreased significantly with the increase of administration concentration.In colorectal cancer HCT15 and SW620 cells treated with AKT agonist(SC-79)alone or in combination with Moxidectin,western blotting showed a decreased proportion of autophagy associated protein LC3II/LC3I.MTT and clonal formation experiments showed that SC-79 combined with Moxidectin significantly promoted cell proliferation compared with Moxidectin experimental group.5.Moxidectin interferes with autophagy by inhibiting CFTR expressionThe TCGA database showed that the expression of CFTR in colorectal cancer tissues was significantly higher than that in normal tissues.Transcriptome sequencing and RT-PCR results of Moxidectin treated colorectal cancer HCT15 cells showed that the expression level of CFTR m RNA in the experimental group was significantly decreased compared with the control group.Histone deacetylase and endoplasmic reticulum stress inhibitor(4-PB)alone or in combination with Moxidectin were used to treat colorectal cancer HCT15 and SW620 cells.Western blotting results showed that compared with moxidectin group,autophagic associated protein p62 and LC3II/LC3I ratio were significantly decreased.MTT and clonal formation experiments showed that4-PB combined with moxidectin significantly increased cell proliferation compared with moxidectin experimental group.Conclusion:Moxidectin inhibits the proliferation of CRC cells and has no obvious effect on cell necrosis and apoptosis,but can affect the expression of CFTR,inhibit the AKT/m TOR pathway to initiate autophagy,damage autophagy flux and interfere with cancer cell proliferation.It provides new insights into the clinical application of Moxidectin.